Recent work from several laboratories has begun to elucidate the molecular basis of several forms of genetic obesity in rodents. This research led to the identification of leptin, a peptide that has a role in regulating appetite, metabolism, and body weight. We have investigated the regulation of leptin secretion by adipose tissue, especially the acute hormonal regulation of leptin secretion. When adipose tissue is incubated in vitro, insulin increases the rate at which leptin is secreted. Furthermore, morphologic studies suggest that insulin alters the subcellular localization of leptin in isolated adipocytes. These changes are consistent with the interpretation that leptin secretion is regulated directly in addition to the action of hormones to regulate transcription of the leptin gene. There are 4 known isoforms of the human leptin receptor (HLR) with different C-terminal cytoplasmic domains. In separate experiments, we have obtained cDNA clones encoding all four isoforms. We have used these reagents to study the intracellular trafficking of these receptors to see if the different tails caused differential targeting of the receptors. We have designated each isoform by the number of unique C-terminal amino acids. As judged by the distribution of leptin binding sites, none of the isoforms were efficiently expressed at the plasma membrane. In cells expressing HLR-67, only 5% of the total leptin binding sites were located at the plasma membrane; in contrast, about 25% of the binding sites were at the plasma membrane in cells expressing HLR-5,-15, or-274. Interestingly, HLR-5 transfected cells expressed 4-fold more total binding sites and thus had more binding sites at the plasma membrane than the other isoforms. Immunofluorescent localization studies showed that all 4 isoforms partially co-localized with calnexin, a marker of the endoplasmic reticulum. All 4 isoforms also partially co- localized with beta-COP (a golgi marker) and were seen in an unidentified punctate compartment. While all the receptors were internalized via clathrin mediated endocytosis, the internalization rates were different, with HLR-15 being internalized the fastest followed by HLR-67, HLR-274, and HLR-5 (in that order). Degradation of internalized leptin was inhibited by leupeptin, indicating that leptin was degraded in lysosomes. Overnight exposure to leptin down-regulated all 4 isoforms, but to a variable extent. HLR-274 displayed the greatest down- regulation and also appeared to reach lysosomes more quickly than the other isoforms. It is noteworthy that HLR- 274 is the isoform that mediates most of the biological actions of leptin, and is also most susceptible to ligand-induced down- regulation.